Cloning and Characterization of the Murine GPI Anchor Synthesis GenePigf,a Homologue of the HumanPIGFGene

Many eukaryotic proteins are bound to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. Its core backbone, which is conserved in different organisms, is synthesized in the endoplasmic reticulum by the sequential addition of glycan components to phosphatidylinositol. One of the human GPI synthesis genes,PIGF(phosphatidylinositol glycan complementation class F), which is involved late in the synthesis pathway, has been cloned. In this study, we isolated complementary and genomic clones ofPigf,a murine counterpart ofPIGF. Pigfencodes a 219 amino acid protein that complements a class F mutation. ThePigfgene consists of six exons spanning 30 kb and was mapped to chromosome 17 at 17E4–E5. These features are very similar toPIGF,thus demonstrating the interspecies conservation of structure, function, gene organization, and genetic locus between these GPI synthesis genes. The results also extend a region in murine distal chromosome 17 that is syntenic to human chromosome 2p16–p22.     

Influence of Axis Removal on Amino-, Carboxy- and Endopeptidase Activities in Cotyledons of Germinating Vigna mungo Seeds

Endopeptidase (azocaseolytic enzyme) and carboxypeptidase activitiesin cotyledons of germinating Vigna mungo seeds increased until3 days after the onset of imbibition and decreased thereafter.In detached and incubated cotyledons, the endopeptidase activityincreased only a little while the carboxypeptidase activitycontinued increasing even after 3 days of incubation. The activitiesof leucine-aminopeptidase and alanine-aminopeptidase, exceptfor that of one leucine-aminopeptidase isoenzyme relativelyabundantly present in unimbibed dry cotyledons, increased slightlyon the first day and declined during germination. In detachedcotyledons, the activities maintained their initial levels throughoutthe incubation period. When cotyledons were detached from germinatingseedlings on days 2 and 4 then incubated, the endopeptidaseactivity started to decrease just after removal of the axisbut the carboxypeptidase activity increased more markedly thanwhen the axis remained attached. Exogenously supplied GA₃, kinetin,IAA, or their combinations, showed no significant effect onthe developmental patterns of the endopeptidase and carboxypeptidaseactivities in cotyledons. These results are discussed in relationto the role of the axis in controlling peptidase formation incotyledons of germinating V. mungo seeds. (Received November 18, 1983; Accepted February 28, 1984)     

Effect of hydrophobic probes on the higher structure of D-amino acid oxidase.

1. The holoenzyme of D-amino acid oxidase [D-amino acid: O2 oxidoreductase (deaminating), EC 1.4.3.3] was found to combine with 1-anilinonaphthalene-8-sulfonate without liberation of its coenzyme, FAD. No energy transfer interaction was found to occur between the bound dye and FAD of the holoenzyme. On the other hand, when the apoenzyme was bound to the dye and then to FAD, energy transfer interaction between the bound dye and bound FAD was observed. In both cases, the dye competes with the substrate, D-alanine. It is concluded that the dye bound to the holoenzyme is oriented in such a special manner that the mutual orientation factor between the dye and FAD becomes very small in magnitude. 2. When the apoenzyme combined with the dye, the monomer-dimer equilibrium of the apoenzyme shifted towards the dimer. On the other hand, 4-monobenzoylamido-4'-aminostilbene-2,2'-disulfonate combined with the apoenzyme to induce monomerization.     

A tryptic peptide from beta-casein depresses protein synthesis and degradation and enhances ureogenesis in primary cultures of rat hepatocytes

1. A peptide which enhances ureogenesis in primary cultured hepatocytes of rats was isolated from a tryptic digest of bovine beta-casein. 2. The structure of the peptide was Ala-Val-Pro-Tyr-Pro-Gln-Arg which is located from 177th to 183rd residues from N-terminal of beta-casein. 3. The peptide also showed the activity to inhibit protein synthesis and protein degradation. 4. It also inhibited DNA synthesis of hepatocytes induced by insulin and/or epidermal growth factor.     

Antigenic analysis of Clostridium chauvoei flagella with protective and non-protective monoclonal antibodies.

Five monoclonal antibodies (mAbs) directed against the flagellin of Clostridium chauvoei were used to analyse the structural and antigenic characteristics on the bacterial flagellar surface. Immune electron microscopy showed that three protective mAbs recognized the surfaced-exposed epitopes on the flagellar filament of this bacteria. In contrast, two non-protective mAbs recognized internal epitopes of the flagellar filament. These findings have been confirmed by ELISA using mAbs absorbed with whole cells of C. chauvoei possessing flagella. Competitive binding assays showed that protective mAbs indicated reciprocal competition, while each of the non-protective mAbs had topographically distinct epitopes. Moreover, immunoblotting analysis with cyanogen-bromide-cleaved flagellin showed that protective mAbs may preferentially recognize conformational epitopes, whilst one of the non-protective mAbs may recognize a linear and conformation-independent epitope in the flagellin of C. chauvoei.     

Alicyclic acid metabolism in plants 8. Association, of two enzymes of the shikimate pathway in shoots of Phaseolus mungo seedlings

The association of two enzymes involved in the shikimate pathway,3-dehydroquinate hydro-lyase (EC 4.2.1.10 [EC] ) and shikimate: NADPoxidoreductase (EC 1.1.1.25 [EC] ), was studied with shoots of etiolated4-day-old Phaseolus mungo seedlings. The enzymes were not separableby ammonium sulfate fractionation, sucrose density gradientcentrifugation, polyacrylamide gel electrophoresis and chromatographyon Sephadex G-100 and DEAE-Sephadex A-50. The results are discussedin relation to the channelling function of metabolites in thealicyclic acid metabolism in higher plants. (Received October 28, 1975; )     

Alicyclic acid metabolism in plants 12. Partial purification and some properties of shikimate kinase from Phaseolus mungo seedlings

Shikimate kinase from Phaseolus mungo seedlings was partiallypurified by DEAEcellulose, hydroxyapatite and Sephacryl S-200column chromatographies. The activity was completely inhibitedby EDTA and the requirement for Mg²⁺ could be partially replacedby Mn²⁺, Ca²⁺; Co²⁺ and Cd²⁺. Sulfhydryl inhibitor did not inhibitthe enzyme activity. The apparent Km values for shikimic acidand ATP at pH 8.6 were 0.25 mM and 0.38 mM, respectively. Theactivity appeared to be maximal at pH 8.6–9.0. Shikimate-3-phosphateand ADP inhibited the activity slightly. Aromatic amino acids,quinic acid and dehydroquinic acid had no significant effecton the activity. (Received January 11, 1979; )     

ADAMTSL6β promotes fibrillin‐1 microfibril assembly,which is possibly mediated via binding through the third thrombospondin type I domain to fibrillin‐1

Fibrillin‐1 is the major component of extracellular matrix microfibrils. Microfibrils dysfunction is responsible for the onset of various connective tissue diseases, including Marfan syndrome. Although ADAMTSL (a disintegrin and metalloproteinase with thrombospondin motifs‐like) 6β is one of the fibrillin‐1 binding proteins, the detailed mechanism underlying the involvement of ADAMTSL6β in microfibril formation remains unclear. In this study, we created deletion mutants of ADAMTSL6β and examined their interactions with fibrillin‐1 assembly. Pull‐down assay of the ADAMTSL6β deletion mutants and fibrillin‐1 protein revealed that ADAMTSL6β binds to fibrillin‐1 through the third thrombospondin type I domain. Furthermore, we observed that formation of fibrillin‐1 matrix assembly was enhanced in MG63 cells, expressing full‐length ADAMTSL6β, when compared with that of wild type MG63 cells. While MG63 cells expressing Δ TSP3‐ADAMTSL6β form showed enhanced assembly formation, Δ TSP2‐ADAMTSL6β form did not enhance that, indicating the difference between Δ TSP2‐Δ TSP3 has a critical role for fibrillin‐1 assembly. As the difference of Δ TSP2‐Δ TSP3 is the third thrombospondin type I domain, we concluded that the third thrombospondin type I domain of ADAMTSL6β influence the microfibril formation. Our data are the functional presentation of the biological role of ADAMTSL6β in the process of microfibril formation.